The purpose of this research proposal is to define the sequence of events that occur as a protein folds to its native conformation from an initially disorganized form. The unique approach described uses asymmetrical, photoactivated bifunctional crosslinking reagents to trap partially folded protein molecules. Protein folding, initiated by a rapid change in environmental conditions, can be coordinated with chemical reactivity of the crosslinking group, initiated by the high flux of photons available from commercial lasers. Subsequent analysis of the peptides crosslinked in this manner by peptide mapping will provide information on the proximity of specific regions of the protein as a function of time following the initiation of folding. Comparison of the results with published x-ray crystallographic structures will show the structural features that arise in specific regions of the protein during folding and chronological order of appearance of these structures. Knowledge of the conformations of partially folded molecules that appear during folding will provide direct evidence for the folding pathway(s) or mechanism(s). The information provided will be useful in predicting tertiary structure from amino acid sequence and therefore is essential to the solution of current problems in medicine, biochemistry, and chemistry. The techniques developed will provide a new method of studying the dynamic aspects of macromolecular structures.